Sleep and wake states are regulated by several brain regions that act via multiple neurotransmitters, neuromodulators and neuropeptides. While several such brain regions and neuropeptides have been identified, additional mechanisms that regulate sleep likely remain to be discovered. Indeed, we recently identified a family of FMRFamide-like neuropeptides that are sufficient to induce a sleep-like state in C. elegans, and found that the zebrafish FMRFamide neuropeptide NPVF is sufficient to induce sleep-like states in both C. elegans and zebrafish. These observations suggest that NPVF acts in an ancient and central mechanism that regulates sleep. While rodents are commonly used for vertebrate sleep studies, genetic tools needed to study NPVF in rodents have not been described. The zebrafish is an alternative vertebrate model that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep, and studies using this simple and inexpensive vertebrate model will provide a rationale to invest the significant time, labor and expense required to generate tools to study NPVF in mice. While the zebrafish has some limitations as a sleep model, its amenability to genetic, optogenetic and pharmacological approaches, as well as its transparency and relatively simple yet conserved vertebrate brain, provide advantages for sleep studies that we exploit in this proposal. Both the protein sequence and hypothalamic expression of NPVF are conserved in zebrafish, rodents and humans, suggesting that findings in zebrafish will be relevant to humans. In Specific Aim 1 we use gain- and loss-of-function genetics to determine whether one or more of the three mature peptides produced by the NPVF preproprotein are necessary and sufficient to promote sleep. We will also determine which receptors mediate the role of NPVF in sleep. In Specific Aim 2 we will test the hypothesis that the ~15 npvf-expressing neurons are necessary and sufficient to promote sleep. We will use noninvasive and high-throughput optogenetic and chemogenetic assays to stimulate, inhibit and ablate NPVF neurons in freely behaving larvae. In Specific Aim 3 we will identify neurons that are activated or inhibited in response to stimulation and inhibition of NPVF neurons using two-photon single plane illumination microscopy and whole-brain calcium imaging. The zebrafish is the only vertebrate model in which whole-brain calcium imaging is currently feasible. This approach will in an unbiased and comprehensive manner identify neurons that may mediate the effects of NPVF on sleep, which we will functionally test using genetics and optogenetics. This project will establish a novel and evolutionarily conserved sleep-regulatory system, and may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neuropsychiatric disorders and may be causal in some cases, this project may also eventually lead to improved therapies for some forms of neuropsychiatric disorders.